Breaking up the correlation between efficacyand toxicity for nonviral gene deliveryMiriam Breunig*, Uta Lungwitz*†, Renate Liebl*, and Achim Goepferich*‡

*Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstrasse 31, 93040 Regensburg, Germany; and †Department of Physicsand Chemistry, University of Southern Denmark-SDU, 5230 Odense, Denmark

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved July 18, 2007 (received for review May 2, 2007)

Nonviral nucleic acid delivery to cells and tissues is considered astandard tool in life science research. However, although an idealdelivery system should have high efficacy and minimal toxicity,existing materials fall short, most of them being either too toxic orlittle effective. We hypothesized that disulfide cross-linked low-molecular-weight (MW) linear poly(ethylene imine) (MW <4.6 kDa)would overcome this limitation. Investigations with these materi-als revealed that the extracellular high MW provided outstandinglyhigh transfection efficacies (up to 69.62 � 4.18% in HEK cells). Weconfirmed that the intracellular reductive degradation producedmainly nontoxic fragments (cell survival 98.69 � 4.79%). When wecompared the polymers in >1,400 individual experiments to sevencommercial transfection reagents in seven different cell lines, wefound highly superior transfection efficacies and substantiallylower toxicities. This renders reductive degradation a highly prom-ising tool for the design of new transfection materials.

biodegradable � polyethylenimine � nucleic acid � disulfide bond �transfection

Gene transfer into cells has become a standard procedure inlife science research. Nonviral carriers have gained in

importance in recent years because of their safety in handlingand ease of application compared with viral vectors. Browsingdatabases such as PubMed or Chemical Abstracts reveals thatalmost every publication related to genetics, biochemistry, mo-lecular biology, developmental biology, or neurology incorpo-rated lipid- or polymer-based transfection as a pivotal tool forthe investigation of various cellular processes. Therefore, itappears that the delivery of nucleic acids is a well establishedmethod for the transfection of mammalian cells, and, given theamount of research data that has been produced, one wouldassume that the procedures for nonviral transfection would bewell optimized. A closer look at the contemporary literature,however, reveals that doubts seem justified. Contemporarytransfection reagents are almost universally toxic because oftheir cationic or amphiphilic character (1–4).

The dilemma that we face is exemplified by considering thepolymeric transfection agent poly(ethylene imine) (PEI). Sinceits introduction in 1995, PEI has been considered the goldstandard for polymer-based gene carriers because of the rela-tively high transfection efficacy of its polyplexes (complex ofnucleic acid and polymer) (5, 6). However, it has been shown thatboth the efficacy and toxicity of PEI are strongly correlated withits molecular weight (MW) as well as its structure (branched orlinear: bPEI or lPEI, respectively) (7–17). Efficacy and adversereactions seem thereby to be strongly associated. PEI is not theonly material that suffers from this ‘‘malignant’’ correlation.Nonviral transfection seems like choosing between Scylla andCharybdis: either a high transfection efficacy, which is associatedwith a devastating toxicity, or a low efficacy, which does notaffect the cell viability at all.

Successful nonviral gene transfection currently requires com-promise to achieve a useful level of transfection efficiency whileminimizing the toxicity. It seems therefore obvious that excessivetoxicity can easily turn nonviral nucleic acid delivery into a

selection process that may render different sets of cell popula-tions of transfected vs. nontransfected and live vs. dead cellsdepending on the reagent in use and may hence make the reagentan undesirably significant factor for the readout of an experi-ment. Surprisingly, little attention has been given in this contextto the fact that toxicity and efficacy are cell type-dependent,because most of the studies describing nonviral transfectionreagents were conducted with only one or two cell types.

One of the goals of our study was to close this gap inknowledge and to concomitantly show that a stringent analyticalmethodology is indispensable to successfully assess efficacies andtoxicities. With our work, we further demonstrate an alternativethat breaks up this correlation between efficacy and toxicity. Wesynthesized polymers that are reductively degradable inside cellsand diminish their charge density to levels that minimize the riskof interactions with cellular components and, therefore, exertsignificantly less toxicity. In contrast to ester- or �-aminoester-linked polyamines, for which hydrolysis half-lives of hours to days(18–23) are a severe handicap, disulfides degrade rapidly notonly inside endolysomes but also to a significant extent in the cellcytoplasm (24, 25), because they do not depend on acid catalysis.To demonstrate the potential of these materials, it was firstnecessary to assess the ability of reductively biodegradablepolymers to condense plasmid DNA into polyplexes that aresuitable for cellular uptake. Furthermore, to confirm the reduc-tively cleavable principle of the polymers, we observed theintracellular trafficking of polyplexes and investigated theirtransfection efficacy in cells with only a low reductive potential.To substantiate our expectations and to give clear instructionsfor the transfection of various cell lines, we evaluated thetransfection efficacy and toxicity of our polymeric transfectionreagents in seven different cell lines and compared them with anumber of commercially available transfection reagents.

Results and DiscussionTo obtain model polymers that allow for reductive degradation,we chose a simple synthesis approach: lPEI 2.6, 3.1, or 4.6 kDawere reversibly cross-linked by dithiodipropionic acid or cystinelinkages to enable the polymer degradation in the presence ofdisulfide reducing agents (Fig. 1). Although intramolecularreactions within PEI molecules are possible, increases in mo-lecular weight [see supporting information (SI) Fig. 7] suggeststhat it is not the dominating mechanism. We obtained a series of

Author contributions: M.B. and U.L. contributed equally to this work; A.G. designedresearch; M.B., U.L., and R.L. performed research; M.B. and U.L. analyzed data; and M.B. andA.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: PEI, poly(ethylene imine); lPEI, linear PEI; bPEI, branched PEI; LRx-lPEIy, lPEIcross-linked with Lomant’s reagent; BCx-lPEIy, lPEI cross-linked with boc-cystine.

‡To whom correspondence should be addressed. E-mail: achim.goepferich@chemie.uni-regensburg.de.

This article contains supporting information online at www.pnas.org/cgi/content/full/0703882104/DC1.

© 2007 by The National Academy of Sciences of the USA

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LRx-lPEIy and BCx-lPEIy polymers [LR and BC refer to thecross-linking reagents, Lomant’s reagent and boc-cystine, re-spectively, x denotes the molar ratio cross-linker/lPEI, and ydenotes the MW (kDa) of the lPEI component (SI Tables 2–4and Fig. 1)]. We cross-linked single lPEI chains to produce abranched structure, which has been shown to more successfullyform small polyplexes with narrow size distribution than thelinear polymer (6). In the following, the results of the mecha-nistic studies are shown for two polymers, namely LR3-lPEI2.6and BC8-lPEI2.6.

As expected, the novel gene carriers complexed plasmid DNAto form nanoparticles in medium of low ionic strength (5%glucose) as well as at physiological salt conditions (150 mMsodium chloride). The polyplexes containing certain cross-linkedlPEIs exhibited hydrodynamic diameters between 600 and 900nm at NP ratios (ratio of nitrogens in polymer to phosphates inDNA) of 6–30 after exposure to serum-free cell culture mediumas determined by laser light scattering (SI Fig. 8).

The capacity of the reductively biodegradable polymers tocondense plasmid DNA into polyplexes that are appropriate forcellular uptake was additionally observed by confocal laserscanning microscopy (CLSM). The first polyplexes were de-tected inside CHO-K1 cells after 30 min of incubation (data notshown), and after 3 h, polyplexes were dispersed throughout thecytosol (Fig. 2A). Analysis of both whole cells and isolated nucleiwith flow cytometry revealed the uptake of fluorescently labeledpolyplexes by �95% of the cells and their corresponding nucleiafter 6 h of incubation, irrespective of which cross-linked poly-mer was used for polyplex formation (Fig. 2B). The cell nucleiwere isolated after incubation with polyplexes by a treatment ofwhole cells with hypotonic buffer after centrifugation at 600 �g. Control experiments with fluorescently labeled DNA onlyshowed neither cellular nor nuclear uptake (data not shown).Although the percentage of cells showing polyplex uptake wasconsistent, the mean fluorescence intensity of whole cells thathad incorporated DNA (and hence the absolute amount ofplasmid DNA inside cells) depended on the polymer used for

polyplex formation (Fig. 2C), but these subtle differences did notcorrespond to differences in the transfection efficiency. Themean fluorescence intensity of nuclei isolated after 6 h ofincubation with polyplexes appeared steady at a value of 50 (Fig.2C; dimensionless value), indicating that an equal amount ofDNA had been transported to the nucleus, irrespective of thepolymer used for polyplex formation. These results suggest thatthe nuclear transport may be the bottleneck in nonviral genedelivery, rather than the cellular uptake.

The disulfide cross-linked lPEIs were designed to exploit thedifferences in the degradation potential at different locationsoutside and inside cells, providing a clear opportunity to designvectors that are stable in the plasma but dissociate within theendolysosomal compartment or the cytoplasm. The concentra-tion of extracellular glutathione (GSH) is usually 100–1,000times less than intracellular GSH and hence favors the mainte-nance of disulfide bonds (24). In our initial experiments, we

Fig. 1. Schematic representation of the synthesis of biodegradable PEIs.LRx-lPEIy (A) and BCx-lPEIy (B) were prepared by cross-linking lPEI with 3,3�-dithiodipropionic acid di(N-succinimidyl ester) or a mixture of N, N�-bis-(tert-butoxycarbonyl)cystine and 4-(4,6-dimethoxy[1.3.5]triazin-2-yl)4-methyl-morpholiniumchlorid hydrate (DMT-MM), respectively, at various molarratios.

Fig. 2. Uptake of polyplexes into CHO-K1 cells. (A) Observation of LR3-lPEI2.6-polyplexes formed at NP 18 in CHO-K1 cells 3 h after transfection byCLSM. The larger green dots (arrow) were polyplex aggregates that were nottaken up by cells. The picture is an overlay of transmitted light and fluores-cence image. (Scale bar, 10 �m.) (B and C) Uptake of polyplexes prepared withYOYO-1-labeled DNA and LR3-lPEI2.6 or BC8-LPEI2.6 in whole cells (■ ) ornuclei (�) after 6 h, as determined by flow cytometry. (B) Fluorescent cells/nuclei with polyplexes indicates the percentage of cells/nuclei that show afluorescence because of intracellular/intranuclear YOYO-1-labeled DNA. (C)The mean fluorescence intensity is represented from those cells or nuclei thathave incorporated YOYO-1-labeled DNA. Statistically significant differencesof pairs are denoted by � (P � 0.01).

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confirmed the destabilization of the cross-linked lPEI-derivedpolyplexes in the presence of disulfide reducing agents, indicat-ing the degradability of the polymer via redox reactions (data notshown). Therefore, we were interested in the response of cross-linked lPEI-mediated gene transfer to changes in the reducing/oxidizing environment of cells. To this end, 1 h before transfec-tion, CHO-K1 cells were treated with 50 �M duroquinone, anagent that oxidizes NADPH/H�, thereby lowering its reducingpotential (26, 27) (see SI Fig. 9). Lower intracellularNADPH/H� levels not only reduced the number of EGFP-positive cells significantly compared with untreated cells (Fig.3A), but the amount of expressed EGFP also declined (Fig. 3B)after both 6 and 24 h. However, the intracellular NADPH/H�

concentration appears to recover with time, because after 24 hthe transfection efficiency was reduced only 1.1- to 1.3-fold ascompared with 1.7-fold after 6 h, depending on the polymer usedfor polyplex formation. When transfecting cells with lowNADPH/H� levels with lPEI polyplexes as a control, the efficacywas not affected at any time point (data not shown). Therefore,from the literature and our results, we conclude that thepolyplexes may at least partially undergo an NADPH/H�-dependent, and therefore GSH-dependent, reduction inside thecell (28–30). The data in Fig. 3 also show that more thantwo-thirds of the transfected cells have already commencedEGFP production after 6 h, a very early time point that has notbeen reported for other polymers so far. Furthermore, the level

of GSH in CHO-K1 cells appeared to be sufficient to activate thecarriers at the tested NP ratios: A pretreatment of CHO-K1 cellswith 1 and 5 mM GSH-monomethylester (GSH MME) toincrease the cellular GSH pool (31) did not improve the geneexpression of polyplexes that were built with biodegradable PEI(see SI Fig. 10).

Observing the intracellular trafficking by CLSM revealed thatmany of the biodegradable PEI-derived polyplexes were colo-calized with acidic organelles after 3 h of incubation in CHO-K1cells (Fig. 4 A and B, arrows). One may expect that this sojournin the lysosomes may result in damage to the transported plasmidDNA. However, by supplementing with 5 mM of sucrose, alysosomotropic agent that accelerates and enhances the escapeof polyplexes from the acidic vesicles (32, 33), we could disprovethis hypothesis. The transfection efficiency with and withoutsucrose was not significantly different (Fig. 4C). Therefore, onecould presume that disulfide bonds begin to degrade in the acidicenvironment of lysosomes, as is described for proteins (34), andthat in the reductive environment of the cytosol, the degradationprocess proceeds.

Fig. 3. Effect of reduced intracellular NADPH/H� concentration, and hencereducing potential, on the transfection efficiency and mean fluorescenceintensity in CHO-K1 cells. Polyplexes were prepared with either LR3-lPEI2.6 (■ )or BC8-lPEI2.6 (�). Duroquinone at a concentration of 50 �M was added to thecells 1 hour before transfection. (A) Values represent the EGFP-positive cells asmeans � SD as determined by flow cytometry 6 h and 24 h after transfection.(B) Values represent the corresponding mean fluorescence intensity of EGFP-positive cells as determined by flow cytometry 6 and 24 h after transfection.Statistically significant differences compared with untreated (without duro-quinone) cells are denoted by ✦ (P � 0.05) or � (P � 0.01).

Fig. 4. Intracellular trafficking of polyplexes. (A and B) Tracking of AlexaFluor 543-labeled plasmid DNA (shown in red) complexed with LR3-lPEI2.6 (A)or BC8-lPEI2.6 (B) cells by CLSM. The acidic organelles of CHO-K1 cells werestained with quinacrine (depicted in turquoise). Most polyplexes were colo-calized with acidic vesicles, some examples are indicated by arrows. The largerred dots were polyplex aggregates that were not taken up by cells. Pictures arean overlay of transmitted light and fluorescence images. (Scale bars, 10 �m.)(C) The effect of the lysosomotropic agent sucrose at 5 mM (white bars) onEGFP expression of polyplexes prepared with LR3-lPEI2.6 or BC8-lPEI2.6 com-pared with transfection without sucrose (black bars) determined by flowcytometry. No statistically significant differences could be found with andwithout sucrose.

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After demonstrating that our cross-linked polymers can beused to form polyplexes, that they are activated in the reductiveenvironment of the cell, are transported to the nucleus, and havethe power to efficiently transfect CHO-K1 cells, we comparedLR3-lPEI2.6 and BC8-lPEI2.6 with the standard polymer-basedtransfection reagents, namely bPEI 25 kDa, lPEI 25 kDa, andlPEI 22 kDa (ExGen) in CHO-K1 cells (Fig. 5). We achieved anoutstanding maximum efficacy of �60% in CHO-K1 cells usingLR3-lPEI2.6 as the gene transfer vehicle, which was by nearly2-fold higher than the best of the polymer standards (ExGen atNP 6 or lPEI 22 kDa at NP 12) (Fig. 5A). Compared with theother polymers, LR3-lPEI2.6 and BC8-lPEI2.6 reached theirmaximum efficacy at relatively high NP ratios (NP 18 and NP 30,respectively). But despite the high polymer amount applied forpolyplex formation, �90% of cells survived the transfectionprocess at all NP ratios (Fig. 5B).

These very promising results prompted us to expand ourtransfection study. We chose various lipid- and polymer-based,commercially available transfection reagents (Table 1) and

compared them with seven of our best cross-linked lPEIs inCHO-K1, COS-7, NIH/3T3, HepG2, HCT116, HeLa, and HEK-293 cells. For each reagent, we evaluated the optimal DNA/reagent ratio in each cell line (for the commercially availablereagents according to the supplier’s protocol), in terms of thetransfection efficiency and cell viability. Altogether, we tested�340 various transfection conditions in the above-mentionedseven cell lines and obtained a detailed knowledge of thetransfection efficacy and corresponding cell viability of the mostcommonly used transfection reagents in comparison with ourcross-linked polymers. The maximal transfection efficiency whencross-linked lPEIs were used as gene delivery vehicles was ashigh or significantly higher than the commercially available

Fig. 6. Comparison of biodegradable PEIs with commercially availabletransfection reagents. (A and B) EGFP-positive cells expressed as maximaltransfection efficiency (A) and corresponding cell viability (B) of variousbiodegradable PEIs and commercially available transfection reagents com-plexed with pEGFP-N1 in (from left to right) CHO-K1 (diagonally hatchedbars), COS-7 (horizontally hatched bars), NIH/3T3 (black bars), HepG2 (hori-zontally striped bars), HCT116 (white bars), HeLa (diamond-hatched bars), andHEK-293 (gray bars) cells as determined by flow cytometry. (C) Transfectionefficiency under conditions where cell viability is �90%. Statistically signifi-cant differences of biodegradable PEIs compared with other transfectionreagents are denoted by ✦ (P � 0.05) or by � (P � 0.01).

Fig. 5. Comparison with standard polymer-based transfection reagents.Transfection efficiency (A) and corresponding cell viability (B) of bPEI 25-kDa(black bars), lPEI 22 kDa (white bars), ExGen (gray bars), LR3-lPEI2.6 (diagonallyhatched bars), and BC8-lPEI2.6 (horizontally hatched bars) complexed withpEGFP-N1 in CHO-K1 cells as determined by flow cytometry. NP ratios at whichbiodegradable PEIs were statistically significant different compared with allother polymers are denoted by � (P � 0.01).

Table 1. Commercially available transfection reagents used in our study

Transfection reagent Supplier Description

PolyFect Qiagen Polycationic dendrimer, branched structureSuperFect Qiagen Polycationic dendrimer, branched structureLipofectamine Invitrogen Liposome formulationTransIT-LT1 Mirus Bio Liposome formulationEffectene Qiagen Nonliposomal lipid formulation with an enhancerFuGENE 6 Roche Blend of lipids in 80% ethanolJetPEI Polyplus Linear PEI 22 kDa

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reagents in six of seven cell lines, reaching efficiencies between40% and 70% (Fig. 6A). In addition, the corresponding cellviability was as high or even significantly higher in three celllines, giving our polymers a clear superiority (Fig. 6B). Only inHepG2 cells was the transfection efficiency �13% higher whena commercially available transfection reagent was used, in thiscase, Lipofectamine (66.23 � 2.23% vs. 53.79 � 2.86%). But thishigher transfection efficacy was also accompanied by a lower cellviability (89.93 � 6.16% vs. 97.21 � 5.56%). The most importantpoint in our study is that if one requires a cell viability �90%after the transfection process, our biodegradable PEIs weregreatly superior to the other lipid- or polymer-based transfectionreagents (Fig. 6C) (for detailed information on which transfec-tion reagent reached the maximal efficiency, please refer to SITable 5). In all cell lines tested, the efficacy was 5- to 7-foldhigher than that of the commercially available reagents whentransfection was conducted under conditions that maintained acell viability �90%. Moreover, in contrast to the biodegradablePEIs, no commercially available transfection reagent was able tomaintain a cell viability of �90% in HeLa cells after thetransfection process.

We have synthesized potentially biodegradable polymers. Weadjusted the degradability to the time scale of cellular events suchas endocytosis. This was achieved by taking advantage of disulfidebonds that are cleaved by redox reactions inside cells. By atransfection screening in different cell lines and a comparison withcommercially available transfection reagents, we identified prom-ising candidates for gene delivery. We conclude that use of thereductive principle in combination with a low-MW polycationicstarting material seems to be a promising strategy for the deliveryof macromolecular, negatively charged substances into cells. Incontrast to other studies that investigated a cross-linking of eitherlow-MW branched (35) or linear (20) polyamines via disulfidebonds, our choice of lPEI 2.6, 3.1 or 4.6 kDa as linear startingmaterial not only entailed a much higher cell viability comparedwith the commercially available standard transfection reagents butalso superior gene transfer efficacy. We could show that it ispossible to achieve a high transfection efficiency (�60%) whilemaintaining a high cell viability. Nonviral transfection no longerrequires choosing between Scylla and Charybdis, and we can hopethat polycationic polymers will indeed find their entry into clinics.

Materials and MethodsSynthesis. lPEI. lPEIs with Mn of 2.6, 3.1, and 4.6 kDa weresynthesized by ring-opening polymerization of 2-ethyl-2-oxazoline (Sigma–Aldrich, Deisenhofen, Germany) and acid-catalyzed hydrolysis of the corresponding poly(2-ethyl-2-oxazoline) as described (36).LRx-lPEIy. Anhydrous lPEI was dissolved in 12 ml of dichlormeth-ane (DCM) and heated to 50°C. The 3,3�-dithiopropionic aciddi-(N-succinimidyl ester) (Lomant’s reagent, LR) (Fluka, Buchs,Switzerland) was dissolved in 5 ml of DCM (specific amountslisted in SI Table 2). The clear solution was added drop-wise tothe lPEI solution under vigorous stirring, and the mixture waskept at 50°C overnight.BCx-lPEIy. Anhydrous lPEI, 4-(4,6-dimethoxy [1.3.5] triazin-2-yl)4-methylmorpholiniumchlorid hydrate (DMT-MM) (Acros Or-ganics, Geel, Belgium), and N, N�-bis-(tert-butoxycarbonyl) cys-tine (boc-cystine, BC) (Advanced Chem Tech, Louisville, KY)were each dissolved in 10 ml, 6 ml, and 4 ml of ethanol,respectively (specific amounts are listed in SI Table 3). The lPEIand BC solutions were transferred into the glass tube of a parallelsynthesis block and mixed by vigorous shaking. The DMT-MMsolution was then added to the clear mixture, and the mixture wasvigorously shaken at room temperature overnight.

For both polymer types, the volatiles were then removed underreduced pressure, the yellow waxen residue was dissolved in 2 MHCl, and the cross-linked lPEI was precipitated with concentrated

aqueous sodium hydroxide. The white gel-like residue was washedwith water until the supernatant became neutral. For analysis, thepurified amine bases were dried at 70°C under reduced pressure.The amine base was transformed into the corresponding hydro-chloride with 2 M HCl to facilitate water solubility.

Formation of Polyplexes. Polyplexes were prepared at NP ratios of6, 12, 18, 24, and 30. Polyplexes were formed by mixing 2 �g ofplasmid DNA (pEGFP-N1) (Clontech, Germany) with the ap-propriate amount of polymer solution, each diluted in 150 mMNaCl. The resulting polyplexes were incubated for 20 min atroom temperature before use. The complexes with commerciallyavailable transfection reagents were prepared according to thesupplier’s protocol.

Functionality of Polymers in Vitro: Transfection and CytotoxicityExperiments. For gene transfer studies, various cell lines (CHO-K1, COS-7, NIH/3T3, HepG2, HCT116, HeLa, and HEK-293)were grown in 24-well plates at an initial density of 38,000–50,000 cells per well. Experiments were performed as describedpreviously and evaluated by flow cytometry using a FACSCali-bur (Becton–Dickinson, Heidelberg, Germany) (37). The cellpopulation referred to the number of whole cells (dead or live)after the transfection process, in contrast to the cellular debris.EGFP-positive cells were detected by using a 515- to 545-nmband-pass filter, whereas the propidium iodide (Sigma–Aldrich)emission was measured with a 670-nm long-pass filter. The meanfluorescence intensity was determined by using the EGFP-positive cells. The transfection efficiency and cell viability werecalculated as follows: First, the cell population of the samples wasnormalized to the cell population of untreated cells as well as thecell viability. In a next step, the number of EGFP-positive orpropidium iodide-negative cells were referred to the calculatedcell population, expressing the transfection efficiency or cellviability, respectively.

Cellular and Nuclear Uptake of Polyplexes. YOYO-1 (MolecularProbes, Eugene, OR) -labeled nucleic acid was used to monitorpolyplex delivery as described (37, 38). Cells were incubatedwith polyplexes for 6 h, followed by f low-cytometry analysis ofwhole cells and nuclei. Brief ly, the nuclei were isolated asfollows: cells were incubated for 5 min in a buffer with low saltconcentration (Tris�HCl 10 mmol/liter, KCl 60 mmol/liter, andEDTA 1 mmol/liter). Thereafter, cells were treated with thesame buffer containing a nonionic detergent (Tergitol TypeNonidet P-40, 0.5%) (Sigma–Aldrich) and protease inhibitors(Roche, Mannheim, Germany). Nuclei were pelleted at 600 �g, rinsed three times with nuclei isolation buffer withoutTergitol Type Nonidet P-40, and then resuspended in PBS. Thepercentage of cells and nuclei that have taken up polyplexesand their mean f luorescence intensity were determined by f lowcytometry after excitation with a 488-nm argon laser anddetection with a 515- to 545-nm band-pass filter.

Intracellular Trafficking: Confocal Laser Scanning Microscopy (CLSM).A Zeiss Axiovert 200 M microscope coupled to a Zeiss LSM 510scanning device (Zeiss, Oberkochen, Germany) was used forCLSM experiments. The inverted microscope was equipped witha Plan-Apochromat �63 objective. Cells were plated in an eight-well Lab-Tek chambered coverglass (Nunc, Wiesbaden, Ger-many), and measurements were directly performed in each wellat 37°C. The thickness of the optical sections was between 0.7and 1.2 �m.

For the investigation of the uptake of polyplexes, YOYO-1-labeled plasmid DNA was used and depicted in green. The fluo-rescent dye was excited with a 488-nm argon laser, and images were

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taken by using a band-pass filter of 505–530 nm in the single-trackmode at the indicated time after the addition of the polyplexes.

For the detection of polyplexes in acidic compartments of thecell, Alexa Fluor 546-labeled plasmid DNA was applied. Poly-plexes and quinacrine mustard (Sigma–Aldrich) at a concentra-tion 10�6 M were added to the cells at the same time. Quinacrinemustard was excited at 458 nm, the fluorescence was imaged byusing a band-pass filter of 475–525 nm, and is depicted inturquoise. Alexa Fluor (Molecular Probes) -labeled DNA wasexcited at 543 nm, and the fluorescence was recorded with a560-nm long-pass filter and is shown in red. A mixture of both

colors indicates a close proximity and therefore an interaction.Images were taken in the multitracking modus.

Statistical Analysis. All measurements were collected (n 3–6)and expressed as means � SD. Single ANOVA was used inconjunction with a multiple comparison test (Tukey test) toassess the statistical significance.

This work was supported, in part, by Deutsche Forschungsgemein-schaft (DFG) Grant BR 3566/1-1. We thank Allison Dennis (GeorgiaInstitute of Technology, Atlanta, GA) for the careful revision of thismanuscript.

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